Alzheimer's disease (AD) is the most common human neurodegenerative disease, leading to cognitive and functional decline and eventual death. In the year 2000, there were 4.5 million AD patients in the United States, accounting for an annual health expenditure of over $83 billion (Cummings, 2004). As life expectancy increases in Western societies, the burden of caring for AD patients is increasing, making management of the disease a leading health priority. Current therapy for AD is mainly directed at improving symptoms, with no possibility of preventing or curing the disease. Available medications include cholinesterase inhibitors to decrease the rate of cognitive decline (Takeda et al, 2006), and psychopharmacological agents to treat psychiatric aspects (Schneider et al, 2003).
Current research is aimed at targeting neuropathological features of AD, in particular, intracellular neurofibrillar tau tangles and extracellular amyloid-β (Aβ) plaques which accumulate in brain tissue of diseased patients. The Aβ peptide, which is cleaved from the amyloid precursor protein (APP), is believed to have an important role in the onset and progression of AD.
Immunization against Aβ has emerged as a promising therapeutic approach for clearing Aβ plaques and reversing cognitive decline. Active immunization using Aβ peptide in various mouse models demonstrated the prevention of AD-like pathology in young animals and attenuation of the disease in older animals (Schenk et al, 1999); prevention of memory loss (Morgan et al, 2000), and reversal of memory deficits (Dodart et al, 2002).
The first clinical trials of immunization in human AD patients involved a vaccine (AN-1792) containing an Aβ1-42 synthetic peptide and the saponin adjuvant QS21. The trial was halted at the Phase II stage due to the development of a transient aseptic meningoencephalitis i.e. an inflammatory response, in ˜6% of the patients (Gilman et al, 2005). The finding of T cell infiltration in brain tissue of affected patients suggested a T cell-mediated autoimmune response (Ferrer et al, 2004).
Despite the cessation of the trial, assessment of a cohort of the participants (30 patients) indicated that 67% of those patients generated antibodies against Aβ, and also showed significantly slower rates of decline of cognitive functions and activities of daily living, as compared to patients without such antibodies. Similar beneficial clinical effects were also present in two of the three patients who had developed immunization-related aseptic meningoencephalitis (Hock et al, 2003). Furthermore, postmortem neuropathological examination of brain tissue of immunized patients showed extensive Aβ plaque clearance in neocortex regions, although tau pathology remained, and T cell meningoencephalitis was evident in at least one patient (Nicoll et al, 2006; Nicoll et al, 2003).
Accordingly, current approaches to optimizing AD immunization are directed to eliminating the detrimental T cell response. Shorter Aβ peptides, containing up to about 15 residues of the N-terminal region have been disclosed to be useful for an AD vaccine, since that region contains the dominant B cell epitopes and substantially lacks T cell epitopes, in both mouse and human (Monsonego et al, 2001; Cribbs et al, 2003). In a mouse model system, Aβ1-11 (i.e. containing the N-terminal 11 amino acids) was found to induce antisera which bound Aβ plaques and triggered plaque clearance (Bard et al, 2003). Furthermore, murine studies showed that the MHC background strongly determines the T cell response to Aβ immunization, and that Aβ-induced encephalitis is mediated by Aβ specific T helper (Th) 1 cells (Monsonego et al, 2006). In middle aged and elderly healthy humans and in AD patients, Aβ-reactive T cell responses are directed against Aβ epitopes located in residues 16-33 (Monsonego et al, 2003).
Various adjuvants or adjuvant sequences have been combined or linked with Aβ peptides, on the basis that the adjuvant stimulates beneficial T helper (Th2) cells required for antibody production. Aβ1-15 covalently coupled to bovine serum albumin induced high titers of anti-Aβ antibodies in immunized APP transgenic mice, while immunization with Aβ1-15 alone was substantially ineffective (Monsonego et al, 2001). Similarly, a prototype AD vaccine containing Aβ1-15 in tandem with the synthetic universal Th cell pan HLA DR epitope (PADRE) produced high titers of anti-Aβ antibodies in immunized mice (Agadjanyan et al, 2005). Use of a second generation vaccine composed of two copies of Aβ1-11 fused to PADRE demonstrated a positive correlation between the concentration of induced antibody and a reduction of cerebral Aβ plaques in immunized transgenic mice with pre-existing AD-like pathology (Petrushina et al, 2007).
Another disclosure of potential AD vaccines relates to four alternative immunogens encompassing either a tandem repeat of two lysine-linked Aβ1-15 sequences (2xAβ1-15) or the Aβ1-15 sequence synthesized to a cross-species T helper cell epitope (T1-Aβ1-15) and each with the addition of a three amino acid Arg-Gly-Asp motif (R-2xAβ1-15; T1-R-Aβ1-15). Intranasal immunization of wild type mice with R-2xAβ1-15 or 2xAβ1-15 plus mutant E. coli heat-labile enterotoxin LT(R192G) adjuvant produced high anti-Aβ antibody titers, and in a murine AD model these vaccines significantly reduced Aβ plaque load. Administration of either T1-Aβ1-15 or T1-R-Aβ1-15 with adjuvant resulted in significantly lower anti-Aβ antibody titers (Maier et al, 2006).
Immunogenic compositions comprising a conjugate composed of a fragment of Aβ linked to a carrier molecule such as diphtheria toxoid, and an adjuvant, are disclosed for example in U.S. Pat. Nos. 6,787,138; 6,946,135 and 6,982,084. Methods of treating a disease characterized by an amyloid deposit of Aβ using such compositions are disclosed for example in U.S. Pat. Nos. 6,866,849; 6,866,850 and 6,787,139.
DNA vaccines against AD have also been described, for example a chimeric DNA minigene encoding Aβ1-42 fused to mouse IL-4 (Ghochikyan et al, 2003); a mouse Aβ1-42 dimer gene for delivery by gene gun, and plasmids encoding Aβ1-42 and Aβ1-16 (Qu et al, 2004); and an adenovirus vector AdPED1-(Aβ1-6)11 encoding 11 tandem repeats of Aβ1-6 (Kim et al, 2007).
HSP60 belongs to a family of chaperone molecules highly conserved throughout evolution, and apparently, no cell can exist without the ability to express HSP60. The human HSP60 molecule was formerly designated HSP65, but was renamed in view of more accurate molecular weight information; as used herein both designations refer to the same protein. Mammalian HSP60 is highly homologous to the bacterial cognates, showing about 50% amino acid identity (Jindal et al, 1989). Thus, HSP60 is shared by the host and its parasites, and is immunogenic, cross-reactive, and universally expressed in inflammation. Furthermore, HSP60 is well recognized by the immune system (Konen-Waisman et al, 1999; Konen-Waisman et al, 1995) and is a part of the set of self-molecules for which autoimmunity naturally exists. Heat shock, IFNγ, bacterial or viral infection, and inflammation, all result in the presentation of endogenous HSP60 epitopes on MHC class II molecules leading to the activation of HSP60-specific T cells, even in healthy individuals (Anderton et al, 1993; Hermann et al, 1991; Koga et al, 1989).
PCT Patent Application No. WO 90/10449, of some of the present inventors, describes a peptide designated p277 having an amino acid sequence corresponding to positions 437-460 of the human HSP65 molecule that is useful as an immunogen inducing resistance to insulin dependent diabetes mellitus (IDDM). A control peptide, designated p278, corresponding to positions 458-474 of human HSP65, did not induce resistance to IDDM.
U.S. Pat. No. 5,736,146, of some of the present inventors, discloses conjugates of poorly immunogenic antigens with a synthetic peptide carrier comprising a T cell epitope derived from the sequence of human heat shock protein HSP65, or an analog thereof, said peptide or analog being capable of increasing substantially the immunogenicity of the poorly immunogenic antigen. According to the disclosure, a peptide corresponding to positions 458-474 and 437-453 of human or mouse HSP60, and homologs thereof, may be conjugated with a wide variety of antigens including peptides, proteins and polysaccharides such as bacterial polysaccharide (e.g. capsular polysaccharide (CPS) Vi of Salmonella typhi), and antigens derived from HIV virus or from malaria antigen.
U.S. Pat. No. 5,869,058, of some of the present inventors, discloses conjugates of poorly immunogenic antigens, e.g., peptides, proteins and polysaccharides, with a synthetic peptide carrier comprising a T cell epitope derived from the sequence of E. coli HSP65 (GroEL), or an analog thereof, said peptide or analog being capable of increasing substantially the immunogenicity of the poorly immunogenic antigen. According to the disclosure, a suitable peptide is Pep278e, which corresponds to positions 437-453 of the E. coli HSP65 molecule.
Barrios et al (1992) disclose that conjugates of 70 kDa HSP or 65 kDa with peptides or oligosaccharides produced high titers of IgG antibodies in immunized mice in the absence of any previous priming with BCG. According to the disclosure, the adjuvant-free carrier effect was T cell dependent, and the use of HSPs as carriers in conjugated constructs was suggested for human vaccine design.
PCT Patent Application No. WO 2006/097914, of some of the present inventors, discloses vaccines comprising an isolated viral antigenic peptide and a synthetic peptide derived from a T cell epitope of HSP60. According to the disclosure, the vaccines include mixtures where the peptide serves as an adjuvant, as well as conjugates where the peptide is covalently linked to the viral antigen. Disclosed peptides include p458, corresponding to positions 458-474 of mouse HSP60, and Ec27, corresponding to positions 391-410 of E. coli HSP60 (GroEL).
There remains an unmet need for a safe and effective vaccine which can prevent and/or attenuate the neuropathology and cognitive decline of Alzheimer's disease. The prior art does not teach or suggest a vaccine comprising an Aβ peptide in combination with a synthetic peptide based on an epitope of HSP60.